Although the polyamines, spermine and spermidine, were originally identified as constituents of seminal fluid and named accordingly, it is well known that these polyamines and their precursor, putrescine, are ubiquitous components of mammalian cells. The enzymatic reactions leading to the formation and interconversion of the polyamines have now been characterized. See, for example, P. McCann et al, eds "Inhibition of Polyamine Metabolism", Academic Press, Inc., Harcourt Brace Jovanovich, New York 1987. These reactions in mammalian cells and protozoa are generally similar to those found in plants and in bacteria, although there are some important differences, such as the absence from mammals of arginine decarboxylase as a route to putrescine via agmatine. Early studies, showing an increased accumulation of putrescine and spermidine in cells during growth and a large and rapid increase in the activity of ornithine decarboxylase (ODC) after application of growth promoting stimuli, suggested that polyamines may play a critical role in cell proliferation. This correlation has now been confirmed more directly in several ways. CHO cell mutants have been obtained which lack functional ODC and these were found to be non-viable in the absence of putrescine or spermidine. Compounds which prevent the synthesis of polyamines have been synthesized and shown to have a profound inhibitory action on cell proliferation. Such inhibitors of polyamine biosynthesis have been searched for and studied intensively over the past 20 years but the major breakthrough in the polyamine field is directly attributable to the synthesis of enzyme-activated irreversible inhibitors of ODC. The availability of these inhibitors has permitted many experiments providing new insight into the function of polyamines and their importance in cellular physiology.
The interconversion and degradation of polyamines is brought about by the action of an intracellular FAD-dependent polyamine oxidase. Its substrates are the N.sup.1 -acetylated derivatives of spermidine and spermine. Therefore, the extent to which these reactions occur is regulated by the activity of PAT which is very highly inducible by an excess of polyamines and by a variety of substances causing cell damage. These reactions allow for the rapid reduction of cellular polyamine concentrations but also permit the reutilization of the putrescine component of the polyamines. This recycling may be of physiological importance since there was a significant decline in tissue spermidine content when N.sup.1,N.sup.4 -bis-(2,3-butadienyl)-1,4-butanediamine, a potent inhibitor of polyamine oxidase, was combined with .alpha.-difluoromethylornithine which inhibits ODC.
Another method of blocking polyamine synthesis has been developed by Porter and colleagues who found that certain synthetic derivatives of polyamines such as N.sup.1,N.sup.8 -bis(ethyl)spermidine and N.sup.1,N.sup.12 -bis(ethyl)spermine mimic the action of their parent polyamines in the repression of ODC and AdoMetDC. Exposure of cells to these compounds therefore leads to the depletion of the natural polyamines. However, these bis(ethyl) analogs will not support growth of L1210 cells and may have some potential as anti-tumor agents and for investigation of the functions of the polyamines which are critical for cell proliferation.
One of the more rewarding areas of recent research in polyamines has been elucidating their role in protozoal growth. The first indication of the impact that parasitic protozoa would have on the polyamine field was the discovery that DMFO would totally cure acute infections of the African trypanosome T. b. brucei in mice. This dramatic finding led to the remarkably swift use of DFMO in what would have been fatal cases of drug resistant late-stage human sleeping sickness in Africa.
A number of the Sporozoea class of protozoa have been shown to be sensitive to the effects of DFMO including Eimeria spp. and the distantly related Pneumocystis carinii. However, some of the most interesting findings have been with the Plasmodia spp., agents of malaria, the most widely distributed and prevalent parasitic disease in the world today. Although original findings from several laboratories have indicated no curative effects of DFMO on erythroacytic forms of malaria in vivo (P. gallinaceum in chickens, and P. berghei in mice), DFMO will block erythrocytic replication (schizogony) in culture in vitro and has recently been shown to significantly reduce parasitemia in P. berghei infections. Interestingly, when exoerythrocytic schizogony (i.e., in liver cells) was studied, DFMO would not only inhibit the cycle in the liver of infected mice, but would also completely inhibit the sporogonous cycle in the mosquito vector. Thus, malaria may well prove to be a significant target for use of inhibitors of polyamine biosynthesis and function.
Applicants have now discovered a novel class of PAO inhibitors.